New methods have been developed for purifying the major viral envelope glycoprotein (gp71) from Rauscher murine leukemia virus (R-MuLV). This purified gp71 was then used to quantitate and characterize the cellular membrane receptors for murine leukemia viruses. The purified gp71 was found to bind specifically to murine cells but not other mammalian cells. Binding was prevented by specific antiserum raised to gp71 (anti-gp71). This binding assay developed can detect receptors as few as 300 murine cells, and with 1 x 10 to the 5th power cells gives significant binding within 30 seconds. The purified glycoprotein retains biologic activity and forms a stable complex with specific receptors on mouse cell membranes. The assay was used to characterize the nature of the cellular receptors that are essential for leukemia virus infection. Purified gp71 binding to mouse cells is prevented if the cells are actively producing related mouse-tropic type C viruses, presumably because the receptors are occupied and are not available to bind exogenously applied gp71. The binding of gp71 to murine cells is enhanced by the presence of calcium ions and low pH. A Scatchard plot showed that there are 700,000 receptors per NIH/3T3 cell, and that the apparent affinity constant (Ka) for their interaction with purified gp71 is 5.6 x 10 to the 10th power Mole-1.